基于生物信息学分析探索PCDHGB4在肺鳞癌发生中的作用

背景与目的 肺鳞状细胞癌（lung squamous cell carcinoma, LUSC）是非小细胞肺癌（non-small cell lung cancer, NSCLC）的亚型之一。有报道原钙黏蛋白γ家族的成员能通过抑制Wnt信号通路来调节肿瘤细胞的生长，原钙黏蛋白γB4（protocadherin-gamma subfamily B4, PCDHGB4）作为家族成员在LUSC中的研究少有报道，本研究旨在通过生物信息学方法探究PCDHGB4在LUSC发生发展中的作用及潜在的预后价值。 方法 应用癌症基因组图谱（The Cancer Genome Atlas, TCGA）、cBioPortal和UALCAN等数据库，对PCDHGB4在LUSC中的表达与预后、临床病理特征、免疫细胞浸润、免疫调节基因、免疫检查点抑制剂（immune checkpoint inhibitors, ICIs）和甲基转移酶等进行分析。单细胞水平的研究对细胞亚型的聚类结果和PCDHGB4在不同免疫细胞亚群中的表达情况进行了分析。此外，我们还比较了LUSC组织与正常组织中PCDHGB4的启动子甲基化水平，并对其进行了蛋白质-蛋白质相互作用和突变分析。最后基于差异表达基因进行富集分析。 结果 生信分析结果显示PCDHGB4在LUSC组织的表达水平低于正常组织。生存分析显示，PCDHGB4表达增加与患者较差的预后有关。单细胞分析显示，PCDHGB4主要在T细胞、单核细胞或巨噬细胞以及树突状细胞中表达，进一步发现PCDHGB4在肿瘤免疫中发挥着不可忽视的作用，并证实了PCDHGB4与免疫检查点途径基因、免疫调节基因和甲基转移酶有一定的相关性。此外，通过富集分析发现PCDHGB4参与了癌症相关的多条通路。 结论 PCDHGB4在LUSC中低表达，PCDHGB4与患者预后不良有关，并且PCDHGB4与肿瘤免疫细胞浸润和通路密切相关。PCDHGB4可能是LUSC潜在的预后标志物和免疫治疗新靶点。

tiple cancer-related pathways.Conclusion The expression of PCDHGB4 was low in LUSC.PCDHGB4 was related to the poor prognosis of patients, and PCDHGB4 was closely related to the infiltration and pathway of tumor immune cells.PCDHGB4 may be a potential prognostic marker and a new target for immunotherapy in LUSC.

Fig 3
Fig 3 Comparison of the prognosis between high and low expression groups of PCDHGB4.A: The prognosis of PCDHGB4 in LUSC; B: The prognosis of PCDHGB4 in NSCLC.A-C: The prognosis of PCDHGB4 in LUSC (OS, DSS, PFS); B: The prognosis of PCDHGB4 in NSCLC (OS, DFS).OS: overall survival; DSS: disease specific survival; PFS: progression-free survival; DFS: disease free survival.

Fig 4 Log
Fig 4 Single cell analysis of PCDHGB4 in LUSC.A: PCDHGB4 aggregation in 40 immune cells; B: Immune cells expressing PCDHGB4; C: Summary of PCDHGB4 expression in 6 single cell datasets; D: Distribution of 6 different cell types and expression level of PCDHGB4 in GSE99254 NSCLC datasets; E: Distribution of 12 different cell types and expression level of PCDHGB4 in GSE127465 NSCLC datasets.

Fig 5
Fig 5 The expression of PCDHGB4 is related to immune cell infiltration.A: The correlation between PCDHGB4 and immune cell infiltration; B: Differences in immune cell infiltration results between PCDHGB4 high and low expression groups; C: The correlation between PCDHGB4 expression and StromalScore, ImmuneScore and ESTIMATEScore in LUSC.aDC: activated DC; iDC: immature DC; pDC: plasmacytoid DC; Tcm: T central memory; Tem: T effector memory; TFH: T follicular helper; Tgd: T gamma delta; Cor: correlation; ns: not significant.*P<0.05;**P<0.01;***P<0.001.

Fig 6
Fig 6 Correlation analysis between PCDHGB4 and immune regulatory genes in LUSC.A: Chemokines, chemokines receptors and MHC genes; B: Immunostimulator genes and immunoinhibitor genes.MHC: major histocompatibility complex.

Fig 8 Fig 9 Fig 10
Fig 8 Relationship of PCDHGB4 with methylation and methyltransferase.A: PCDHGB4 promoter methylation level in LUSC; B: PCDHGB4 promoter methylation level in LUSC tissues at different tumor stages; C: Heatmap of DNA methylation at the CpG site of the PCDHGB4 gene from the MethSurv database; D: Correlation between PCDHGB4 expression and m1A, m5C and m6A regulatory genes.*P<0.05.
Tab 1 Clinicopathological features of LUSC patients with low and high expression of PCDHGB4 M1 4 (2.0%) 2 (1.1%)Data acquisition: Download and sort out RNAseq data of STAR process of TCGA-LUSC project from TCGA database and extract TPM format data and clinical data; Data filtering strategy: Remove normal+remove no clinical information; Missing value processing: Remove samples with missing variables; Data processing method: log2(value+1); TNM: tumor-node-metastasis; TCGA: The Cancer Genome Atlas.
Download and sort out RNAseq data of STAR process of TCGA-LUSC project from TCGA database and extract TPM format data and clinical data.Data filtering strategy: remove normal+remove no clinical information.Missing value processing: Variable missing is not uniformly processed (Pathologic N stage: 6 cases; Pathologic stage: 4 cases; Age: 6 cases; Smoker: 12 cases).Data processing method: log2(value+1).